Transient (about 2 hr) acidification to approx. pH 5.0 of agar-gelled overlayers containing untransformed NRK-49F or KiMSV-transformed NRK-49F cells in the presence of fetal calf serum or crude 49F-cell conditioned medium, as sources of latent TGF-beta, elicited EGF-dependent colony formation of 49F cells and inhibited spontaneous growth of transformed cells. Pure, active TGF-beta (porcine, type I) had the same effects on these respective cell types, suggesting that the above results were due to activation of latent TGF-beta in the transiently acidic cellular environment. Similar acidifications in the absence of a source of latent TGF-beta enhanced the positive growth response of 49F and AKR-2B cells to EGF and active TGF-beta and also the negative growth response of KiMSV-transformed 49F cells to active TGF-beta. These results are compatible with the idea that acidic cellular environments, particularly in tumor tissues, are conducive to activation of latent TGF-beta, perhaps in conjunction with other activating mechanisms, and to an enhanced response to some growth factors. However, the heterogeneity of cell populations within tumoral masses presents an obstacle to a clear understanding of the consequences of such activation.